Pib v5 his topo ta vector

We chose four exemplar species, one from each taxonomic family, for downstream analysis: A. asperrimus, P. schultei, R. artemis, and S. sipylus. We designed gene-specific forward and reverse primers (Table S1) to amplify the complete ORF of each putative enzyme from the cDNA, and cloned them into Top10 cells with the pIB/V5-His TOPO/TA^® vector (Invitrogen). We included Kozak sequences (RCCATGG) at the 3′ end of the forward primers and did not include the stop codon in the reverse primers. Colony PCR or direct sequencing was done to ensure the genes were cloned in the correct direction, then we extracted the plasmids with a GeneJET™ Plasmid Miniprep Kit (Thermo Scientific) and transfected them into Sf9 cells (Invitrogen) using the reagent FuGENE HD (Promega). Culture medium was harvested after 72 hours incubation at 27 °C and centrifuged, and the supernatant tested for successful expression via Western Blot (Figure S1) with anti-V5-HRP antibody (Invitrogen). Plate assays for substrate activity were performed on the individual enzymes following the same protocol as the whole gut extracts.

The cDNAs encoding arylsulfataselike genes of P. xylostella (PxGSS1, PxGSS2, PxGSS3, and PxSulfD), Bombyx mori SulfC1 (BmC1), and Yponomeuta cagnagella C (YcC) were amplified by PCR using gene-specific primers (supplementary table S1, Supplementary Material online), which included a 5′ Kozak sequence and lacked the native stop codon to facilitate epitope and His-tag fusion expression. PCR products were ligated into a pIB/V5-His TOPO TA vector (Invitrogen), and correct sequence and orientation were verified by Sanger sequencing. Sf9 cells were cultivated in GIBCO Sf-900 II SFM (Invitrogen) on six-well plates at 27 °C until 70–90% confluence was achieved. Transfection was performed with FuGENE HD (Promega) following the manufacturer’s protocol. At 72 h after transfection, the culture medium of Sf9 cells was harvested and aliquots were directly used for blotting or for activity assays. Expressed proteins were detected with an anti-V5 HRP antibody (Thermo Fisher Scientific) using the SuperSignal West HisProbe Kit (Pierce).